Golgi-to-ER retrograde transport prevents premature differentiation of Drosophila type II neuroblasts via Notch-signal-sending daughter cells.

Stem cells are heterogeneous to generate diverse differentiated cell types required for organogenesis; however, the underlying mechanisms that differently maintain these heterogeneous stem cells are not well understood. In this study, we identify that Golgi-to-endoplasmic reticulum (ER) retrograde transport specifically maintains type II neuroblasts (NBs) through the Notch signaling. We reveal that intermediate neural progenitors (INPs), immediate daughter cells of type II NBs, provide Delta and function as the NB niche. The Delta used by INPs is mainly produced by NBs and asymmetrically distributed to INPs. Blocking retrograde transport leads to a decrease in INP number, which reduces Notch activity and results in the premature differentiation of type II NBs. Furthermore, the reduction of Delta could suppress tumor formation caused by type II NBs. Our results highlight the crosstalk between Golgi-to-ER retrograde transport, Notch signaling, stem cell niche, and fusion as an essential step in maintaining the self-renewal of type II NB lineage.

Study on the Mechanism of Capillary Leakage Caused by Hypoxia-Inducible Factor-1α through Inducing High Expression of Matrix Metalloproteinase-9.

PURPOSES: This study mainly explored the mechanism of capillary leakage caused by hypoxia-inducible factor-1α through inducing high expression of matrix metalloproteinase-9. Method. We established a monolayer endothelial cell model by culturing human umbilical vein endothelial cells (HUVEC) in vitro, used tumor necrosis factor (TNFα) and HIF-1α inhibitor 2-methoxyestradiol (2ME2) to act on HUVEC, and at the same time constructed siRNA-transfected HUVEC to interfere with the expression of HIF-1α. The permeability of monolayer endothelial cells was measured by transwell chamber method, the concentration of MMP-9 in the supernatant was measured by ELISA method, the expression of key molecules related to permeability (HIF- 1α, MMP-9, claudin-5, and ZO-1) was measured by RT-PCR and Western blot method, and the localization and expression of claudin-5 and ZO-1 were measured by immunofluorescence method. We searched for 7 HIF-1α hypoxia response elements within 4000 bp before the transcription start site in the MMP-9 promoter region, constructed the MMP-9 promoter-luciferase reporter gene recombinant plasmid, transfected and stimulated HUVEC with TNFα, and detected the effect of 7 hypoxia response element plasmids on the transcription activity of MMP-9 promoter. RESULTS: Under the action of TNFα, the permeability of monolayer endothelial cells increased, and the concentration of MMP-9 in the cell supernatant increased. 2ME2 and HIF-1α-siRNA transfection can improve the above situation (P < 0.05). 2ME2 and HIF-1α-siRNA transfection can inhibit the high expression of HIF-1α and MMP-9 caused by TNFα, thereby increasing the expression of claudin-5 and ZO-1 (P < 0.05). 2ME2 and HIF-1α-siRNA transfection can reduce the inhibition of TNFα on the expression of cell membrane protein claudin-5 and tight junction protein ZO-1. Element 1, element 5, and element 7 are the sites where HIF-1α interacts with MMP-9 at the transcription level. CONCLUSION: This study shows that HIF-1α can increase the permeability of monolayer epithelial cells by inducing the high expression of MMP-9, leading to capillary leakage. Its target is at the -3798 bp, -1878 bp, and -1489 bp points of the transcription initiation site in the MMP-9 promoter region.

Molecular Characterization of Hamster-Adapted Yellow Fever Virus.

We previously reported two hamster models for viscerotropic yellow fever virus (YFV) infection: one using a YFV strain (Jiménez), isolated from a fatal human case in Panama in 1974, and the other using the prototype YFV strain (Asibi). Asibi hamster passage 7 (P7) was associated with accumulation of seven amino acid substitutions, including five in the envelope protein. In this study we report the genome sequences of the hamster Jiménez P0 and P10 viruses in which we identified only two amino acid substitutions during passage, one each in the nonstructural proteins NS3 and NS5, indicating a role for the nonstructural proteins in increased YFV viscerotropism in the Jiménez hamster model. Thus, there are multiple molecular mechanisms involved in viscerotropism of YFV in the hamster model. Neither Asibi P7 nor Jiménez P10 viruses were viscerotropic in mice or guinea pigs. Thus, the hamster viscerotropic phenotype did not translate to other laboratory rodent species.

Effect of probiotics on the incidence of ventilator-associated pneumonia in critically ill patients: a randomized controlled multicenter trial.

PURPOSE: To evaluate the potential preventive effect of probiotics on ventilator-associated pneumonia (VAP). METHODS: This was an open-label, randomized, controlled multicenter trial involving 235 critically ill adult patients who were expected to receive mechanical ventilation for ≥48 h. The patients were randomized to receive (1) a probiotics capsule containing live Bacillus subtilis and Enterococcus faecalis (Medilac-S) 0.5 g three times daily through a nasogastric feeding tube plus standard preventive strategies or (2) standard preventive strategies alone, for a maximum of 14 days. The development of VAP was evaluated daily, and throat swabs and gastric aspirate were cultured at baseline and once or twice weekly thereafter. RESULTS: The incidence of microbiologically confirmed VAP in the probiotics group was significantly lower than that in the control patients (36.4 vs. 50.4 %, respectively; P = 0.031). The mean time to develop VAP was significantly longer in the probiotics group than in the control group (10.4 vs. 7.5 days, respectively; P = 0.022). The proportion of patients with acquisition of gastric colonization of potentially pathogenic microorganisms (PPMOs) was lower in the probiotics group (24 %) than the control group (44 %) (P = 0.004). However, the proportion of patients with eradication PPMO colonization on both sites of the oropharynx and stomach were not significantly different between the two groups. The administration of probiotics did not result in any improvement in the incidence of clinically suspected VAP, antimicrobial consumption, duration of mechanical ventilation, mortality and length of hospital stay. CONCLUSION: Therapy with the probiotic bacteria B. Subtilis and E. faecalis are an effective and safe means for preventing VAP and the acquisition of PPMO colonization in the stomach.

[Combination of Weighted index of comorbidities and sepsis-related organ failure assessment score in death risk evaluation of septic patients].

OBJECTIVE: To predict the risk of 28- day mortality on septic patients in intensive care unit (ICU) with the combination of the Weighted index of comorbidities (WIC) and sepsis-related organ failure assessment (SOFA) score. METHODS: The clinical data of adult sever sepsis/ septic shock patients in Department of Emergency Medicine of Chagzeng Hospital and Department of Clinical Care Medicine of Jinan Military General Hospital from October 2011 to February 2013 were analyzed retrospectively. The etiological factor, past history, having sever sepsis or not were recorded. Age score, WIC score, acute physiology and chronic health evaluation II (APACHE II) score and SOFA score were calculated at or 24 hours after admission. The logistic regression was used and the receiver operating characteristic curve (ROC curve) was drawn to calculate the patients' outcome. RESULTS: In 310 enrolled patients, 223 (71.9%) patients survived and 87 (28.1%) died. Univariate analysis showed the P values of the age score, WIC score, APACHE II score and SOFA score. chronic cardiac insufficiency, type 2 diabetes, cerebrovascular disease, tumor, multiple injury, pulmonary infection and having severe sepsis or not were all less tha 0.2. The above 11 variables were put into the multivariate logistic regression equation 1, of which predicted probability was reserved. It revealed that 5 variables were independently associated with 28-day prognosis, of which influence power in descending order were SOFA score [odds ratio (OR)=1.308, 95 % confidence interval (95% CI): 1.158-1.478, P=0.000], having sever sepsis or not (OR=0.206, 95% Cl:0.100-0.424, P=0.000), APACHE II score (OR=1.090, 95% CI:1.021-1.164, P=0.010) WIC score (OR=1.441, 95% CI:1.067-1.947, P=0.017) age score (OR=1.228, 95% CI:1.027-1.468, P=0.024), the Wals were 18.554, 18.369, 6.725, 5.662, 5.067, respectively. The 3 variables, age score, WIC score and SOFA score, were brought into the multivariate logistic regression equation 2, of which predicted probability was reserved too. It revealed that age score (OR=1.330, 95 % CI: 1.145-1.546, P=0.000), WIC score (OR=1.496, 95% CI: 1.145-1.546, P=000) and SOFA score (OR=1.429, 95% CI: 1.303-1.567, P=0.000), were independently associated with the septic patients' 28-day prognosis. There was no significant difference in the area under receiver operating characteristics curve (AUC) between the SOFA score and APACHE II score (0.784 vs. 0.780, Z=0.014, P=0.989). However, compared with APACHE II score, the AUC of equation 1 (0.888) and 2 (0.851) were much more (Z=4.333, P= 0.000; Z= 2.669, P= 0.008). CONCLUSION: The sensitivity of 28-day prognosis 28-day prognosis by WIC score was improved greatly with the combination of SOFA score and age score.

Role of BC loop residues in structure, function and antigenicity of the West Nile virus envelope protein receptor-binding domain III.

Site-directed mutagenesis of residues in the BC loop (residues 329-333) of the envelope (E) protein domain III in a West Nile virus (WNV) infectious clone and in plasmids encoding recombinant WNV and dengue type 2 virus domain III proteins demonstrated a critical role for residues in this loop in the function and antigenicity of the E protein. This included a strict requirement for the tyrosine at residue 329 of WNV for virus viability and E domain III folding. The absence of an equivalent residue in this region of yellow fever group viruses and most tick-borne flavivirus suggests there is an evolutionary divergence in the molecular mechanisms of domain III folding employed by different flaviviruses.

Development of resistance to passive therapy with a potently neutralizing humanized monoclonal antibody against West Nile virus.

Previous studies have established the therapeutic efficacy of humanized E16 (hE16) monoclonal antibody against West Nile virus in animals. Here, we assess the potential for West Nile virus strains encoding mutations in the hE16 epitope to resist passive immunotherapy and for the selection of neutralization escape variants during hE16 treatment. Resistance to hE16 in vivo was less common than expected, because several mutations that affected neutralization in vitro did not significantly affect protection in mice. Moreover, the emergence of resistant variants after infection with fully sensitive virus occurred but was relatively rare, even in highly immunocompromised B and T cell-deficient RAG mice.

Genetic variation of St. Louis encephalitis virus.

St. Louis encephalitis virus (SLEV) has been regularly isolated throughout the Americas since 1933. Previous phylogenetic studies involving 62 isolates have defined seven major lineages (I-VII), further divided into 14 clades. In this study, 28 strains isolated in Texas in 1991 and 2001-2003, and three older, previously unsequenced strains from Jamaica and California were sequenced over the envelope protein gene. The inclusion of these new sequences, and others published since 2001, has allowed better delineation of the previously published SLEV lineages, in particular the clades of lineage II. Phylogenetic analysis of 106 isolates identified 13 clades. All 1991 and 2001-2003 isolates from Nueces, Jefferson and Harris Counties (Texas Gulf Coast) group in clade IIB with other isolates from these counties isolated during the 1980s and 1990s. This lack of evidence for introduction of novel strains into the Texas Gulf Coast over a long period of time is consistent with overwintering of SLEV in this region. Two El Paso isolates, both from 2002, group in clade VA with recent Californian isolates from 1998-2001 and some South American strains with a broad temporal range. Overall, these data are consistent with multiple introductions of SLEV from South America into North America, and provide support for the hypothesis that in most situations, SLEV circulates within a locality, with occasional incursions from other areas. Finally, SLEV has much lower nucleotide (10.1 %) and amino acid variation (2.8 %) than other members of the Japanese encephalitis virus complex (maximum variation 24.6 % nucleotide and 11.8 % amino acid).

A combination of naturally occurring mutations in North American West Nile virus nonstructural protein genes and in the 3' untranslated region alters virus phenotype.

We previously reported mutations in North American West Nile viruses (WNVs) with a small-plaque (sp), temperature-sensitive (ts), and/or mouse-attenuated (att) phenotype. Using an infectious clone, site-directed mutations and 3' untranslated region (3'UTR) exchanges were introduced into the WNV NY99 genome. Characterization of mutants demonstrated that a combination of mutations involving the NS4B protein (E249G) together with either a mutation in the NS5 protein (A804V) or three mutations in the 3'UTR (A10596G, C10774U, A10799G) produced sp, ts, and/or att variants. These results suggested that the discovery of North American WNV-phenotypic variants is rare because of the apparent requirement of concurrent polygenic mutations.

A mutation in the envelope protein fusion loop attenuates mouse neuroinvasiveness of the NY99 strain of West Nile virus.

Substitutions were engineered individually and in combinations at the fusion loop, receptor-binding domain and a stem-helix structure of the envelope protein of a West Nile virus strain, NY99, and their effects on mouse virulence and presentation of epitopes recognized by monoclonal antibodies (MAbs) were assessed. A single substitution within the fusion loop (L107F) attenuated mouse neuroinvasiveness of NY99. No substitutions attenuated NY99 neurovirulence. The L107F mutation also abolished binding of a non-neutralizing MAb, 3D9, whose epitope had not been previously identified. MAb 3D9 was subsequently shown to be broadly cross-reactive with other flaviviruses, consistent with binding near the highly conserved fusion loop.

A single amino acid substitution in the central portion of the West Nile virus NS4B protein confers a highly attenuated phenotype in mice.

West Nile virus (WNV) NS4B is a small hydrophobic nonstructural protein that is hypothesized to participate both in viral replication and evasion of host innate immune defenses. The protein has four cysteine residues (residues 102, 120, 227, and 237). Since cysteines are often critical for the function of proteins, each of the four cysteine residues found in WNV NS4B was mutated to serine by site-directed mutagenesis. While three of these substitutions had little effect on replication or mouse virulence phenotypes, the C102S mutation was associated with a temperature-sensitive phenotype at 41 degrees C as well as attenuation of the neuroinvasive and neurovirulence phenotypes in mice.

Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains.

The introduction of West Nile virus (WNV) into North America has been associated with relatively high rates of neurological disease and death in humans, birds, horses, and some other animals. Previous studies identified strains in both genetic lineage 1 and genetic lineage 2, including North American isolates of lineage 1, that were highly virulent in a mouse neuroinvasion model, while other strains were avirulent or significantly attenuated (D. W. C. Beasley, L. Li, M. T. Suderman, and A. D. T. Barrett, Virology 296:17-23, 2002). To begin to elucidate the basis for these differences, we compared a highly virulent New York 1999 (NY99) isolate with a related Old World lineage 1 strain, An4766 (ETH76a), which is attenuated for mouse neuroinvasion. Genomic sequencing of ETH76a revealed a relatively small number of nucleotide (5.1%) and amino acid (0.6%) differences compared with NY99. These differences were located throughout the genome and included five amino acid differences in the envelope protein gene. Substitution of premembrane and envelope genes of ETH76a into a NY99 infectious clone backbone yielded a virus with altered in vitro growth characteristics and a mouse virulence phenotype comparable to ETH76a. Further site-specific mutagenesis studies revealed that the altered phenotype was primarily mediated via loss of envelope protein glycosylation and that this was associated with altered stability of the virion at mildly acidic pH. Therefore, the enhanced virulence of North American WNV strains compared with other Old World lineage 1 strains is at least partly mediated by envelope protein glycosylation.

A rapid and noninvasive method for detecting tissue-limited mosaicism: detection of i(12)(p10) in buccal smear from a child with Pallister-Killian syndrome.

Pallister-Killian syndrome (PKS), a rare disorder, is characterized by tissue-limited or tissue-specific mosaicism. The characteristic chromosome abnormality associated with PKS is i(12p), which is seen predominantly in skin fibroblast cultures. Diagnosis of i(12p) has been carried out on buccal smears before and was shown to be an easy and feasible method. All previously published studies used alpha-satellite probes for the diagnosis and as such have several pitfalls. Our approach, using dual-color, locus-specific probes, has high specificity and sensitivity for the diagnosis of i(12p). Using statistical analysis, we have also confirmed that the signal pattern in interphase nuclei is consistent with isochromosome 12p.

Prenatal diagnosis of a fetus with unbalanced translocation (4;13)(p16;q32) with overlapping features of Patau and Wolf-Hirschhorn syndromes.

Wolf-Hirschhorn syndrome (WHS) and Patau syndrome are two of the most severe conditions resulting from chromosome abnormalities. WHS is caused by a deletion of 4p16, while Patau syndrome is caused by trisomy for some or all regions of chromosome 13. Though the etiologies of these syndromes differ, they share several features including pre- and postnatal growth retardation, microcephaly, cleft lip and palate, and cardiac anomalies. We present here a female fetus with deletion of 4p16 --> pter and duplication of 13q32 --> qter due to unbalanced segregation of t(4;13)(p16;q32) in the father. She displayed overlapping features of both of these syndromes on ultrasound. To the best of our knowledge, this is the first report of a fetus with both partial trisomy 13 and deletion of 4p16, the critical region for WHS.